The role of recombination activating gene (RAG) reinduction in thymocyte development in vivo

Assembly of T cell receptor (TCR)alpha/beta genes by variable/diversity/joining (V[D]J) rearrangement is an ordered process beginning with recombination activating gene (RAG) expression and TCR beta recombination in CD4(-)CD8(-)CD25(+) thymocytes. In these cells, TCR beta expression leads to clonal expansion, RAG downregulation, and TCR beta allelic exclusion. At the subsequent CD4(+)CD8(+) stage, RAG expression is reinduced and V(D)J recombination is initiated at the TCR alpha locus. This second wave of RAG expression is terminated upon expression of a positively selected alpha/beta TCR. To examine the physiologic role of the second wave of RAG expression, we analyzed mice that cannot reinduce RAG expression in CD4(+)CD8(+) T cells because the transgenic locus that directs RAG1 and RAG2 expression in these mice is missing a distal regulatory element essential for reinduction. In the absence of RAG reinduction we find normal numbers of CD4(+)CD8(+) cells but a 50-70% reduction in the number of mature CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes. TCR alpha rearrangement is restricted to the 5' end of the J alpha cluster and there is little apparent secondary TCR alpha recombination. Comparison of the TCR alpha genes expressed in wild-type or mutant mice shows that 65% of all alpha/beta T cells carry receptors that are normally assembled by secondary TCR alpha rearrangement. We conclude that RAG reinduction in CD4(+)CD8(+) thymocytes is not required for initial TCR alpha recombination but is essential for secondary TCR alpha recombination and that the majority of TCR alpha chains expressed in mature T cells are products of secondary recombination.